scholarly journals Bovine Liver Chromatin Fraction Contains Actin Polymerization Activity Inducing Micronuclei Formation when Injected into Prometaphase Cultured Cells

2002 ◽  
Vol 27 (1) ◽  
pp. 39-45 ◽  
Author(s):  
Yosuke Matsuoka ◽  
Yuriko Matsuoka ◽  
Satoshi Shibata ◽  
Yoshihiro Yoneda
Keyword(s):  
Actin Polymerization ◽  
Cultured Cells ◽  
Bovine Liver ◽  
Chromatin Fraction ◽  
Liver Chromatin

scholarly journals Arp2/3 and Unc45 maintain heterochromatin stability in Drosophila polytene chromosomes

2019 ◽  
Author(s):  
George Dialynas ◽  
Laetitia Delabaere ◽  
Irene Chiolo
Keyword(s):  
Actin Filaments ◽  
Genome Stability ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Nuclear Periphery ◽  
Polytene Chromosomes ◽  
Nuclear Actin ◽  
Nuclear Dynamics ◽  
Ectopic Recombination ◽  
Repair Pathways

AbstractRepairing DNA double-strand breaks (DSBs) is particularly challenging in pericentromeric heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination. InDrosophilaKc cells, accurate homologous recombination (HR) repair of heterochromatic DSBs relies on the relocalization of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. This movement is driven by Arp2/3-dependent nuclear actin filaments and myosins’ ability to walk along them. Conserved mechanisms enable the relocalization of heterochromatic DSBs in mouse cells, and their defects lead to massive ectopic recombination in heterochromatin and chromosome rearrangements. InDrosophilapolytene chromosomes, extensive DNA movement is blocked by a stiff structure of chromosome bundles. Repair pathways in this context are poorly characterized, and whether heterochromatic DSBs relocalize in these cells is unknown. Here, we show that damage in heterochromatin results in relaxation of the heterochromatic chromocenter, consistent with a dynamic response in this structure. Arp2/3, the Arp2/3 activator Scar, and the myosin activator Unc45, are required for heterochromatin stability in polytene cells, suggesting that relocalization enables heterochromatin repair in this tissue. Together, these studies reveal critical roles for actin polymerization and myosin motors in heterochromatin repair and genome stability across different organisms and tissue types.Impact StatementHeterochromatin relies on dedicated pathways for ‘safe’ recombinational repair. In mouse and fly cultured cells, DNA repair requires the movement of repair sites away from the heterochromatin ‘domain’vianuclear actin filaments and myosins. Here, we explore the importance of these pathways inDrosophilasalivary gland cells, which feature a stiff bundle of endoreduplicated polytene chromosomes. Repair pathways in polytene chromosomes are largely obscure and how nuclear dynamics operate in this context is unknown. We show that heterochromatin relaxes in response to damage, and relocalization pathways are necessary for repair and stability of heterochromatic sequences. This deepens our understanding of repair mechanisms in polytenes, revealing unexpected dynamics. It also provides a first understanding of nuclear dynamics responding to replication damage or rDNA breaks, providing a new understanding of the importance of the nucleoskeleton in genome stability. We expect these discoveries to shed light on tumorigenic processes, including therapy-induced cancer relapses.

Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization

2010 ◽  
Vol 29 (10) ◽  
pp. 1139-1145 ◽  
Author(s):  
Kairo Wakamatsu ◽  
Masaru Fujimoto ◽  
Mikio Nakazono ◽  
Shin-ichi Arimura ◽  
Nobuhiro Tsutsumi
Keyword(s):  
Actin Polymerization ◽  
Cultured Cells ◽  
Atp Level ◽  
Suspension Cultured Cells

Aldolase-localization in cultured cells: Cell-type and substrate-specific regulation of cytoskeletal associations

2001 ◽  
Vol 79 (6) ◽  
pp. 719-728 ◽  
Author(s):  
Ralf Schindler ◽  
Elke Weichselsdorfer ◽  
Oliver Wagner ◽  
Jürgen Bereiter-Hahn
Keyword(s):  
Electron Microscopy ◽  
Intermediate Filaments ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Actin Binding ◽  
Permeabilized Cells ◽  
Specific Regulation ◽  
Actin Fibrils ◽  
Fructose 1,6 Bisphosphate

The role of aldolase as a true F- and G-actin binding protein, including modulating actin polymerization, initiating bundling, and giving rise to supramolecular structures that emanate from actin fibrils, has been established using indirect immunofluorescence, permeabilization of XTH-2 cells and keratocytes, and microinjection of fluorescence-labeled aldolase. In addition, binding to intermediate filaments, vimentin, and cytokeratins has been demonstrated. In permeabilized cells in the presence of fructose-1,6-bisphosphate (20–2000 µM) aldolase shifts from association with actin fibres to intermediate filaments. Plenty of free binding sites on microtubules have been revealed by addition of fluorochromed aldolase derived from rabbit skeletal muscle. However, endogenous aldolase was never found associated with microtubules. Differences in actin polymerization in the presence of aldolase as revealed by pyrene-labeled actin fluorimetry and viscosimetry were explained by electron microscopy showing the formation of rod-like structures (10 nm wide, 20–60 nm in length) by association of aldolase with G-actin, which prevents further polymerization. Upon the addition of fructose-1,6-bisphosphate, G-actin–aldolase mixture polymerizes to a higher viscosity and forms stiffer filaments than pure actin of the same concentration.Key words: aldolase, cytoskeleton, electron microscopy, viscosimetry.

scholarly journals The Arf-GEF Steppke promotes F-actin accumulation, cell protrusions and tissue sealing during Drosophila dorsal closure

2020 ◽  
Author(s):  
Junior J. West ◽  
Tony J. C. Harris
Keyword(s):  
Actin Polymerization ◽  
Cultured Cells ◽  
Genetic Interaction ◽  
Coiled Coil ◽  
Leading Edge ◽  
Cell Junctions ◽  
Drosophila Embryo ◽  
Dorsal Closure ◽  
Ph Domain ◽  
Actin Networks

AbstractCytohesin Arf-GEFs promote actin polymerization and protrusions of cultured cells, whereas the Drosophila cytohesin, Steppke, antagonizes actomyosin networks in several developmental contexts. To reconcile these findings, we analyzed epidermal leading edge actin networks during Drosophila embryo dorsal closure. Here, Steppke is required for F-actin of the actomyosin cable and for actin-based protrusions. steppke mutant defects in the leading edge actin networks are associated with improper sealing of the dorsal midline, but are distinguishable from effects of myosin mis-regulation. Steppke localizes to leading edge cell-cell junctions with accumulations of the F-actin regulator Enabled emanating from either side. Enabled requires Steppke for full leading edge recruitment, and genetic interaction shows the proteins cooperate for dorsal closure. Steppke over-expression induces ectopic, actin-rich, lamellar cell protrusions, an effect dependent on the Arf-GEF activity and PH domain of Steppke, but independent of Steppke recruitment to myosin-rich AJs via its coiled-coil domain. Thus, Steppke promotes actin polymerization and cell protrusions, effects that occur in conjunction with Steppke’s previously reported regulation of myosin contractility during dorsal closure.

scholarly journals Indispensable functions of ABL and PDGF receptor kinases in epithelial adherence of attaching/effacing pathogens under physiological conditions

2014 ◽  
Vol 307 (2) ◽  
pp. C180-C189 ◽  
Author(s):  
Carolin F. Manthey ◽  
Christine B. Calabio ◽  
Anna Wosinski ◽  
Elaine M. Hanson ◽  
Bruce A. Vallance ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Actin Polymerization ◽  
Intestinal Inflammation ◽  
Cultured Cells ◽  
Kinase Inhibitor ◽  
P38 Map Kinase ◽  
Bacterial Attachment ◽  
Infection Model ◽  
Pdgf Receptor

Enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium are attaching-and-effacing (A/E) pathogens that cause intestinal inflammation and diarrhea. The bacteria adhere to the intestinal epithelium, destroy microvilli, and induce actin-filled membranous pedestals but do not invade the mucosa. Adherence leads to activation of several host cell kinases, including FYN, n-SRC, YES, ABL, and ARG, phosphorylation of the bacterial translocated intimin receptor, and actin polymerization and pedestal formation in cultured cells. However, marked functional redundancy appears to exist between kinases, and their physiological importance in A/E pathogen infections has remained unclear. To address this question, we employed a novel dynamic in vitro infection model that mimics transient and short-term interactions in the intestinal tract. Screening of a kinase inhibitor library and RNA interference experiments in vitro revealed that ABL and platelet-derived growth factor (PDGF) receptor (PDGFR) kinases, as well as p38 MAP kinase, have unique, indispensable roles in early attachment of EPEC to epithelial cells under dynamic infection conditions. Studies with mutant EPEC showed that the attachment functions of ABL and PDGFR were independent of the intimin receptor but required bacterial bundle-forming pili. Furthermore, inhibition of ABL and PDGFR with imatinib protected against infection of mice with modest loads of C. rodentium, whereas the kinases were dispensable for high inocula or late after infection. These results indicate that ABL and PDGFR have indispensable roles in early A/E pathogen attachment to intestinal epithelial cells and for in vivo infection with limiting inocula but are not required for late intimate bacterial attachment or high inoculum infections.

scholarly journals Proteins regulating actin assembly in oogenesis and early embryogenesis of Xenopus laevis: gelsolin is the major cytoplasmic actin-binding protein.

1988 ◽  
Vol 107 (4) ◽  
pp. 1489-1498 ◽  
Author(s):  
T Ankenbauer ◽  
J A Kleinschmidt ◽  
J Vandekerckhove ◽  
W W Franke
Keyword(s):  
Xenopus Laevis ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Early Embryogenesis ◽  
Actin Binding ◽  
Dependent Manner ◽  
Expression Library ◽  
Cytoplasmic Actin ◽  
Carboxy Terminal

Oocytes, notably those of amphibia, accumulate large pools of nonfilamentous ("soluble") actin, both in the cytoplasm and in the nucleoplasm, which coexist with extensive actin filament arrays in the cytoplasmic cortex. Because the regulation of oogenically accumulated actin is important in various processes of oogenesis, egg formation, fertilization and early embryogenesis, we have purified and characterized the major actin-binding proteins present in oocytes of Xenopus laevis. Here we report that the major actin-binding component in the ooplasm, but not in the nucleus, is a polypeptide of Mr approximately 93,000 on SDS-PAGE that reduces actin polymerization in vitro in a Ca2+-dependent manner but promotes nucleation events, and also reduces the viscosity of actin polymers, indicative of severing activity. We have raised antibodies against the purified oocyte protein and show that it is different from villin, is also prominent in unfertilized eggs and early embryos and is very similar to a corresponding protein present in various tissues and in cultured cells, and appears to be spread over the cytoplasm. Using these antibodies we have isolated a cDNA clone from a lambda gt11 expression library of ovarian poly(A)+-RNA. Determination of the amino acid sequence derived from the nucleotide sequence, together with the directly determined sequence of the amino terminus of the native protein, has shown that this clone encodes the carboxy-terminal half of gelsolin. We conclude that gelsolin is the major actin-modulating protein in oogenesis and early embryogenesis of amphibia, and probably also of other species, that probably also plays an important role in the various Ca2+-dependent gelation and contractility processes characteristic of these development stages.

scholarly journals Manganese promotes intracellular accumulation of AQP2 via modulating F-actin polymerization and reduces urinary concentration in mice

2018 ◽  
Vol 314 (2) ◽  
pp. F306-F316 ◽  
Author(s):  
Lei Lei ◽  
Ming Huang ◽  
Limin Su ◽  
Dongping Xie ◽  
Fahmy A. Mamuya ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Cultured Cells ◽  
Critical Role ◽  
Water Channel ◽  
Urine Concentration ◽  
Urinary Concentration ◽  
Intracellular Accumulation ◽  
Rhoa Activity ◽  
Cytoskeletal Dynamics

Aquaporin-2 (AQP2) is a water channel protein expressed in principal cells (PCs) of the kidney collecting ducts (CDs) and plays a critical role in mediating water reabsorption and urine concentration. AQP2 undergoes both regulated trafficking mediated by vasopressin (VP) and constitutive recycling, which is independent of VP. For both pathways, actin cytoskeletal dynamics is a key determinant of AQP2 trafficking. We report here that manganese chloride (MnCl2) is a novel and potent regulator of AQP2 trafficking in cultured cells and in the kidney. MnCl2 treatment promoted internalization and intracellular accumulation of AQP2. The effect of MnCl2 on the intracellular accumulation of AQP2 was associated with activation of RhoA and actin polymerization without modification of AQP2 phosphorylation. Although the level of total and phosphorylated AQP2 did not change, MnCl2 treatment impeded VP-induced phosphorylation of AQP2 at its serine-256, -264, and -269 residues and dephosphorylation at serine 261. In addition, MnCl2 significantly promoted F-actin polymerization along with downregulation of RhoA activity and prevented VP-induced membrane accumulation of AQP2. Finally, MnCl2 treatment in mice resulted in significant polyuria and reduced urinary concentration, likely due to intracellular relocation of AQP2 in the PCs of kidney CDs. More importantly, the reduced urinary concentration caused by MnCl2 treatment in animals was not corrected by VP. In summary, our study identified a novel effect of MnCl2 on AQP2 trafficking through modifying RhoA activity and actin polymerization and uncovered its potent impact on water diuresis in vivo.

scholarly journals Tir Triggers Expression of CXCL1 in Enterocytes and Neutrophil Recruitment during Citrobacter rodentium Infection

2015 ◽  
Vol 83 (9) ◽  
pp. 3342-3354 ◽  
Author(s):  
Valerie F. Crepin ◽  
Maryam Habibzay ◽  
Izabela Glegola-Madejska ◽  
Marianne Guenot ◽  
James W. Collins ◽  
...  
Keyword(s):  
Amino Acids ◽  
Immune Responses ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Citrobacter Rodentium ◽  
Lesion Formation ◽  
Direct Role ◽  
Content Type ◽  
Host Immune Responses ◽  
Mucosal Surfaces

The hallmarks of enteropathogenicEscherichia coli(EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogenCitrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 inC. rodentiumcolonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses toC. rodentiuminfection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that aC. rodentiumstrain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected withC. rodentiumexpressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses toC. rodentiuminfection.

scholarly journals The Arf-GEF Steppke promotes F-actin accumulation, cell protrusions and tissue sealing during Drosophila dorsal closure

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0239357
Author(s):  
Junior J. West ◽  
Tony J. C. Harris
Keyword(s):  
Actin Polymerization ◽  
Cultured Cells ◽  
Genetic Interaction ◽  
Coiled Coil ◽  
Leading Edge ◽  
Cell Junctions ◽  
Dorsal Closure ◽  
Ph Domain ◽  
Dorsal Midline ◽  
Actin Networks

Cytohesin Arf-GEFs promote actin polymerization and protrusions of cultured cells, whereas the Drosophila cytohesin, Steppke, antagonizes actomyosin networks in several developmental contexts. To reconcile these findings, we analyzed epidermal leading edge actin networks during Drosophila embryo dorsal closure. Here, Steppke is required for F-actin of the actomyosin cable and for actin-based protrusions. steppke mutant defects in the leading edge actin networks are associated with improper sealing of the dorsal midline, but are distinguishable from effects of myosin mis-regulation. Steppke localizes to leading edge cell-cell junctions with accumulations of the F-actin regulator Enabled emanating from either side. Enabled requires Steppke for full leading edge recruitment, and genetic interaction shows the proteins cooperate for dorsal closure. Inversely, Steppke over-expression induces ectopic, actin-rich, lamellar cell protrusions, an effect dependent on the Arf-GEF activity and PH domain of Steppke, but independent of Steppke recruitment to myosin-rich AJs via its coiled-coil domain. Thus, Steppke promotes actin polymerization and cell protrusions, effects that occur in conjunction with Steppke’s previously reported regulation of myosin contractility during dorsal closure.

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